Malaria is responsible for hundreds of millions of cases and a million deaths annually. An ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents all disease and transmission. When radiation attenuated Pf sporozoites (Pf SPZ) are administered by bite of infected mosquitoes, > 90% of human volunteers are protected for at least 10 months against experimental Pf challenge. Sanaria's goal is to develop and commercialize an attenuated Pf SPZ vaccine that prevents Pf blood stage infection in > 90% of recipients. Such a vaccine has the potential for > $1 billion annual revenues in markets in the developed and developing world. Sanaria has succeeded in establishing robust, reproducible, and consistent manufacture and release of clinical lots of its radiation attenuated PfSPZ Vaccine, received FDA approval for its IND, and in the 2nd quarter of 2009 will initiate a Phase 1 clinical trial to assess safety and protective efficacy of the PfSPZ Vaccine. After demonstrating safety, the goal is to move rapidly to safety and proof of concept efficacy studies, expanded Phase 2 studies and Phase 3 studies in Africa to support licensure. There is a clear path to taking this PfSPZ Vaccine to registration and commercialization. However, a number of experts believe that it would be preferable if the SPZ were attenuated by permanent genetic alteration (i.e. gene deletions) rather than by irradiation. This elegant, defined approach has the potential to be safer and might give rise to more potent attenuated SPZ. For our Phase I project, the goal was to produce at least one genetically modified Pf knock out (KO) clone that produced Pf SPZ that could 1) invade human hepatocytes, 2) develop in hepatocytes and produce proteins not expressed in SPZ, and 3) not be able to fully mature in hepatocytes (i.e. be fully attenuated). Our collaborative work has led to production of two single KO clones (p52 and fabI) that fulfill the 1st two goals, one of which (p52) has already been shown to fulfill all three, and a double KO clone (lacking p52 and p36) that has yet to be characterized. The single KO clones were created by single site crossover disruption and all three retain a selectable marker. Herein, we propose a systematic approach that will lead to the production of at least one Pf parasite clone optimized for attenuation, safety, potency, and suitability for manufacture. This involves deleting selected pairs of genes, each resulting in liver stage attenuation, using a double crossover strategy. This approach will maximize the likelihood of sustained attenuation and safety. Removal of selectable markers will eliminate the chance of inducing antibiotic resistance in the host or the environment. Testing of multiple doubly attenuated KO clones will maximize the prospects of identifying one that is optimal for gametocyte and SPZ production, attenuation, and potency. Process development and an engineering production campaign will produce genetically attenuated SPZ that fulfill regulatory requirements and can be manufactured and then tested in humans as a candidate vaccine. PUBLIC HEALTH RELEVANCE: A highly effective malaria vaccine would have an enormous public health benefit. Sanaria's attenuated malaria sporozoite vaccine (PfSPZ Vaccine) is expected to be highly effective and will be studied in clinical trials in 2009. The PfSPZ Vaccine is attenuated (weakened) by irradiation of each vaccine lot. This research aims to eliminate the need for irradiation in manufacturing a PfSPZ vaccine, by creating parasites that are attenuated by gene removal.